Specificity protein 1 is a novel target of 2, 4-bis (p-hydroxyphenyl)-2-butenal for the suppression of human oral squamous cell carcinoma cell growth

Background

The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects though 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. The purpose of this study was to investigate the anti-proliferative effects of 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) on two oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, through regulation of specificity protein 1 (Sp1).

Results

HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies. Sp1 was significantly inhibited by HPB242 in a dose-dependent manner. Furthermore, cell cycle regulating proteins and anti-apoptotic proteins, which are known as Sp1 target genes, were altered at the molecular levels. The key important regulators in the cell cycle such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, myeloid leukemia sequence 1 (Mcl-1) and survivin were significantly decreased by HPB242 or suppressed Sp1 levels, however pro-apoptotic proteins caspase3 and PARP were cleaved in HN22 and HSC4.

Conclusions

HPB242 may be useful as a chemotherapeutic agent for OSCC for the purpose of treatment and prevention of oral cancer and for the improvement of clinical outcomes.     

Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo

Purpose: Carrimycin is a newly synthesized macrolide antibiotic with good antibacterial effect. Exploratory experiments found its function in regulating cell physiology, proliferation and immunity, suggesting its potential anti-tumor capacity. The aim of this study is to investigate the anti-tumor effect of carrimycin against human oral squamous cell carcinoma cells in vitro and in vivo.Methods: Human oral squamous cell carcinoma cells (HN30/HN6/Cal27/HB96 cell lines) were treated with gradient concentration of carrimycin. Cell proliferation, colony formation and migration ability were analyzed. Cell cycle and apoptosis were assessed by flow cytometry. The effect of carrimycin on OSCC in vivo was investigated in tumor xenograft models. Immunohistochemistry, western blot assay and TUNEL assays of tissue samples from xenografts were performed. The key proteins in PI3K/AKT/mTOR pathway and MAPK pathway were examined by western blot.Results: As the concentration of carrimycin increased, the proliferation, colony formation and migration ability of OSCC cells were inhibited. After treating with carrimycin, cell cycle was arrested in G0/G1 phase and cell apoptosis was promoted. The tumor growth of xenografts was significantly suppressed. Furthermore, the expression of p-PI3K, p-AKT, p-mTOR, p-S6K, p-4EBP1, p-ERK and p-p38 were down-regulated in vitro and in vivo.Conclusions: Carrimycin can inhibit the biological activities of OSCC cells in vitro and in vivo, and regulate the PI3K/AKT/mTOR and MAPK pathways.     

Hispolon induces apoptosis in oral squamous cell carcinoma cells through JNK/HO-1 pathway activation

Oral squamous cell carcinoma (OSCC) has a high recurrence rate and poor prognosis. Hispolon, a polyphenolic compound with antiviral, antioxidant, and anticancer activities, is a potential chemotherapy agent. However, few studies have investigated the anti-cancer mechanism of hispolon in oral cancer. This present study used the cell viability assay, clonogenic assay, fluorescent nuclear staining, and flow cytometry assay to analyse the apoptosis-inducing effects of hispolon in OSCC cells. After hispolon treatment, the apoptotic initiators, cleaved caspase-3, −8, and − 9, were upregulated, whereas the cellular inhibitor of apoptosis protein-1 (cIAP1) was downregulated. Furthermore, a proteome profile analysis using a human apoptosis array revealed the overexpression of heme oxygenase-1 (HO-1) by hispolon, which was determined to be involved in caspase-dependent apoptosis. Moreover, cotreatment with hispolon and mitogen-activated protein kinase (MAPK) inhibitors revealed that hispolon induces apoptosis in OSCC cells through activation of the c-Jun N-terminal kinase (JNK) pathway and not the extracellular signal-regulated kinase (ERK) or p38 pathway. These findings indicate that hispolon may exert an anticancer effect on oral cancer cells by upregulating HO-1 and inducing caspase-dependent apoptosis by activating the JNK pathway.     

p21活化激酶1在口腔癌细胞侵袭和迁移中的作用

p21活化激酶1(PAK1)与多种癌症类型的进展有关,然而,其在口腔癌中的作用仍不明确。本研究以人舌鳞状细胞癌(oral squamous cell carcinoma,OSCC)HSC4细胞系为研究材料,考察了PAK1在细胞系中的定位及血清生长因子对PAK1定位的影响。通过转染PAK1 siRNA来敲低OSCC细胞中的PAK1,并分析PAK1敲低对细胞侵袭和迁移性的影响。研究发现PAK1在人舌鳞状细胞癌细胞系HSC4中主要定位于细胞核,而血清生长因子可调节PAK1在不同亚细胞区域中的易位或积累,并且还可能影响OSCC细胞的细胞骨架结构。此外,敲低PAK1可显著抑制OSCC细胞的迁移和侵袭能力,表明PAK1在OSCC发生发展过程中起着至关重要的作用。     

Pristimerin induces apoptosis and tumor inhibition of oral squamous cell carcinoma through activating ROS-dependent ER stress/Noxa pathway

BackgroundPristimerin (Pri), a natural quinone methide triterpenoid isolated from Celastraceae and Hippocrateaceae, exhibits potent antitumor activity against various cancers. However, the mechanism of apoptosis induction by Pri in oral squamous cell carcinoma (OSCC) and its anti-OSCC effect in vivo has not been widely studied.PurposeThis study aimed to investigate the anti-OSCC activities of Pri in vitro and in vivo and addressed the potential mechanisms of Pri-induced apoptosis.MethodsThe effects of Pri on OSCC cells were analyzed by cell viability, colony formation and flow cytometry assays. Western blotting and qRT-PCR assays were chosen to detect the expression of proteins and genes. The anti-OSCC efficacy of Pri in vivo was evaluated by CAL-27 xenografts.ResultsWe showed that Pri inhibited the proliferation of human OSCC cell lines. Additionally, Pri induced apoptosis by upregulating Noxa expression. Furthermore, Pri treatment triggered excessive endoplasmic reticulum (ER) stress activation and subsequently induced c-Jun N-terminal kinase (JNK) signaling. ROS scavengers and ER stress inhibitors significantly attenuated Pri-induced OSCC cell apoptosis. Finally, Pri suppressed tumor growth in CAL-27 xenografts, accompanied ER stress activation and cell apoptosis.ConclusionThese results reveal that Pri suppressed tumor growth and triggered cell apoptosis through ER stress activation in OSCC cells and xenografts, suggesting that Pri may serve as a therapeutic agent for OSCC.     

Long non‐coding RNA highly up‐regulated in liver cancer promotes epithelial‐to‐mesenchymal transition process in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is an oral and maxillofacial malignancy that exhibits high incidence worldwide. In diverse human cancers, the long non‐coding RNA (lncRNA) highly up‐regulated in liver cancer (HULC) is aberrantly expressed, but how HULC affects OSCC development and progression has remained mostly unknown. We report that HULC was abnormally up‐regulated in oral cancer tissues and OSCC cell lines, and that suppression of HULC expression in OSCC cells not only inhibited the proliferation, drug tolerance, migration and invasion of the cancer cells, but also increased their apoptosis rate. Notably, in a mouse xenograft model, HULC depletion reduced tumorigenicity and inhibited the epithelial‐to‐mesenchymal transition process. Collectively, our findings reveal a crucial role of the lncRNA HULC in regulating oral cancer carcinogenesis and tumour progression, and thus suggest that HULC could serve as a novel therapeutic target for OSCC.     

CircKRT1 drives tumor progression and immune evasion in oral squamous cell carcinoma by sponging miR-495-3p to regulate PDL1 expression

Regulatory functions of circRNAs by targeting the micro RNA (miRNA)/mRNA axis have been increasingly found in oral squamous cell carcinoma (OSCC). CircRNA keratin 1 (CircKRT1) and miR-495-3p were dysregulated in OSCC. Programmed death ligand 1 (PDL1) was an important immunotherapeutic molecule in OSCC. Our objective was to explore whether circKRT1 could regulate cancer progression and immune evasion in OSCC by affecting the miR-495-3p/PDL1 axis. RNA expression was examined by quantitative real-time polymerase chain reaction. All protein levels were detected by western blot. OSCC cell growth was assessed by CCK-8 and colony formation assays. Cell migratory and invasive abilities were evaluated by transwell assay. CD8⁺ T-cell cytotoxicity was determined via lactate dehydrogenase assay. CD8⁺ T-cell percentage and apoptosis were analyzed by flow cytometry. Target screening was performed by Veen Diagram and RNA pull-down assay. Target binding was verified using dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft in mice was conducted for in vivo experiment. CircKRT1 and PDL1 were highly expressed in OSCC tissues and cells. CircKRT1 knockdown repressed OSCC cell growth, migration, invasion, epithelial–mesenchymal transition, and CD8⁺ T-cell apoptosis, but enhanced CD8⁺ T cytotoxicity and percentage. The inhibitory effects of circKRT1 downregulation on OSCC progression and immune evasion were related to PDL1 expression inhibition. CircKRT1 sponged miR-495-3p and miR-495-3p targeted PDL1. OSCC progression and immune evasion were regulated by circKRT1 via the miR-495-3p/PDL1 axis. CircKRT1 also facilitated OSCC progression in vivo by regulating miR-495-3p and PDL1. This study clarified that circKRT1 worked as a miR-495-3p sponge to regulate PDL1, consequently affecting cancer progression and immune evasion in OSCC.     

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound,inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies

Background

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, might possess anti-cancer properties. We aimed to evaluate the efficacy of Riccardin D-26 as a candidate compound for treatment of cancers with sensitive or drug resistant cells.

Methods

Experiments were performed on human oral squamous carcinoma KB cells and vincristin-selected MDR KB/VCR cells. The inhibition of cell growth was evaluated by colorimetric and clonogenic assays. The apoptotic cells were determined by the Annexin V-FITC/PI staining assay. JC-1 fluorescence probe was used to examine the mitochondria membrane potential (MMP). Further experiments were performed in nude mice bearing KB or KB/VCR xenografts. Riccardin D-26 was administered by injection for 2 weeks. The specimens of KB and KB/VCR xenografts were removed for TUNEL staining and Western blotting analysis.

Results

Riccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26's activity in cancer cells was greater than that in human normal liver cells. In mice, Riccardin D-26 effectively prevented the growth of KB and KB/VCR xenografts without significant toxicity. Further studies suggested that Riccardin D-26 inhibited cancer growth by inducing apoptosis in the activation of mitochondria-mediated intrinsic apoptosis pathway. Riccardin D-26 also possessed this activity in regulation of mitogen-related protein kinases such as MAPK and PI3K/Akt, which is associated with its inhibitory effect on KB/VCR cells.

Conclusions

Riccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells.

General significance

Riccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.     

A Novel Compound NSC745885 Exerts an Anti-Tumor Effect on Tongue Cancer SAS Cells In Vitro and In Vivo

Objective

Oral squamous cell carcinoma (OSCC) is a prevalent cancer, especially in developing countries. Anthracyclines and their anthraquinone derivatives, such as doxorubicin, exhibit a cell growth inhibitory effect and have been used as anti-cancer drugs for many years. However, the cardiotoxicity of anthracycline antibiotics is a major concern in their clinical application. NSC745885 is a novel compound synthesized from 1,2-diaminoanthraquinone, which subsequently reacts with thionyl chloride and triethylamine. The present study aimed to investigate the anti-oral cancer potential and the safety of NSC745885.

Methods

We investigated the anti-cancer potential of NSC745885 in oral squamous carcinoma cell lines and in an in vivo oral cancer xenograft mouse model. The expression of apoptotic related genes were evaluated by real-time RT-PCR and western bloting, and the in vivo assessment of apoptotic marker were measured by immunohistochemical staining. The anti-tumor efficiency and safety between doxorubicin and NSC745885 were also compared.

Results

Our results demonstrated that NSC745885 exhibits anti-oral cancer activity through the induction of apoptosis in cancer cells and in tumor-bearing mice, and this treatment did not induce marked toxicity in experimental mice. This compound also exhibits a comparable anti-tumor efficiency and a higher safety in experimental mice when compared to doxorubicin.

Conclusions

The data of this study provide evidence for NSC745885 as a potential novel therapeutic drug for the treatment of human OSCC.     

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.     

Nuclear survivin promoted by acetylation is associated with the aggressive phenotype of oral squamous cell carcinoma

Defects in apoptotic pathway contribute to development and progression of oral cancer. Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is increased in many types of cancers. However, it is unclear whether increased survivin is associated with oral squamous cell carcinomas (OSCC), and what mechanisms may involve in. In this study, we examined survivin expression in OSCC compared with normal oral tissues via immunohistochemical staining. The results showed that, not only total survivin is increased in OSCCs, but also the subcellular location of survivin is changed in OSCCs compared with normal oral tissues. In most of normal oral tissues, survivin staining was either negative, or cytoplasmic positive/nuclear negative; whereas in most of OSCC tissues, survivin staining was nuclear positive. Statistic analysis indicates that nuclear survivin, rather than total or cytoplasmic one, correlates with tumor TNM stage and differentiation grade. Consistently, in vitro analysis showed that survivin is in cytoplasm in normal human oral kinotinocyte (HOK) cells; whereas it is in nucleus in OSCC HN6 cells. Importantly, treatment of HOK cells with HDAC inhibitor Trichostatin A (TSA) induces survivin acetylation and promotes its nuclear localization. Moreover, nuclear survivin in OSCC cells was acetylated at K129 in its C-terminal, suggesting that the acetylation is important for nuclear location of survivin. Our study demonstrates that it is nuclear survivin, rather than total or cytoplasmic one, associates with TNM stage and tumor grade of OSCC. Thus, we propose nuclear survivin as a prognostic marker for the progression of OSCC.     

SL-01, an oral derivative of gemcitabine,inhibited human breast cancer growth through induction of apoptosis

SL-01 is an oral derivative of gemcitabine that was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl) pyrazine-2-carbonyl at N4-position on cytidine ring of gemcitabine. We aimed to evaluate the efficacy of SL-01 on human breast cancer growth. SL-01 significantly inhibited MCF-7 proliferation as estimated by colorimetric assay. Flow cytometry assay indicated the apoptotic induction and cell cycle arrest in G1 phase. SL-01 modulated the expressions of p-ATM, p53 and p21 and decrease of cyclin D1 in MCF-7 cells. Further experiments were performed in a MCF-7 xenografts mouse model. SL-01 by oral administration strongly inhibited MCF-7 xenografts growth. This effect of SL-01 might arise from its roles in the induction of apoptosis. Immunohistochemistry assay showed the increase of TUNEL staining cells. Western blotting indicated the modulation of apoptotic proteins in SL-01-treated xenografts. During the course of study, there was no evidence of toxicity to mice. In contrast, the decrease of neutrophil cells in peripheral and increase of AST and ALT levels in serum were observed in the gemcitabine-treated mice. Conclusion: SL-01 possessed similar activity against human breast cancer growth with gemcitabine, whereas, with lower toxicity to gemcitabine. SL-01 is a potent oral agent that may supplant the use of gemcitabine.     

p38 and ERK MAP kinase mediates iron chelator-induced apoptosis and -suppressed differentiation of immortalized and malignant human oral keratinocytes

Iron is essential for neoplastic cell growth, and iron chelators have been tested for potential anti-proliferative and anti-cancer effects, but the effects of iron chelators on oral cancer have not been clearly elucidated. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during iron chelator-induced apoptosis and differentiation of immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). The iron chelator deferoxamine (DFO) exerted potent time- and dose-dependent inhibitory effects on the growth and apoptosis of IHOK and HN4 cells. DFO strongly activates p38 MAP kinase and extracellular signal-regulated kinase (ERK), but does not activate c-Jun N-terminal kinase/stress-activated protein kinase. Of the three MAP kinase blockers used, the selective p38 MAP kinase inhibitor SB203580 and ERK inhibitor PD98059 protected IHOK and HN4 cells against iron chelator-induced cell death, which indicates that the p38 and ERK MAP kinase is a major mediator of apoptosis induced by this iron chelator. Interestingly, treatment of IHOK and HN4 cells with SB203580 and PD98059 abolished cytochrome c release, as well as the activation of caspase-3 and caspase-8. DFO suppressed the expression of epithelial differentiation markers such as involucrin, CK6, and CK19, and this suppression was blocked by p38 and ERK MAP kinase inhibitors. Collectively, these data suggested that p38 and ERK MAP kinase plays an important role in iron chelator-mediated cell death and in the suppression of differentiation of oral immortalized and malignant keratinocytes, by activating a downstream apoptotic cascade that executes the cell death pathway.     

Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF‐κB pathway

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration‐dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF‐κB p65, MMP‐2, and MMP‐9. Ectopic expression of constitutively active NF‐κB p65 prevented platycodin D‐mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC‐4 xenograft tumors and reduced phosphorylation of NF‐κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF‐κB pathway and might provide therapeutic benefits in the treatment of OSCC.     

Hyaluronic acid engrafted metformin loaded graphene oxide nanoparticle as CD44 targeted anti-cancer therapy for triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence.MethodsUV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model.ResultsHylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinβ1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor.ConclusionsHA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo.General significanceHA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.     

RNA-Mediated Gene Silencing of Nicotinamide N-Methyltransferase Is Associated with Decreased Tumorigenicity in Human Oral Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.